3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a colorimetric assay for assessing cell metabolic activity. It is a tetrazolium-based compound that is reduced by metabolically active cells, converting the yellow MTT into purple formazan crystals.

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6 protocols using 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt

Peptide Synthesis and Cell Culture Protocols

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In this experimental study, Fmoc (9-fluorenylmethoxycarbonyl)-
protected amino acids with
various protected side chains, rink-amide-MBHA
resin, and O-(benzotriazole-1-yl)-N, N, N´,
N´-tetramethyluronium-hexafluorophosphate
(HBTU) were purchased from GL Biochem
(Shanghai, China). HPLC grade trifluoroacetic
acid (TFA), acetonitrile (ACN) and water, N-ethyldiisopropylamine
(DIEA) and triisopropylsilane
(TIPS) were products of Merck Chemicals (Germany).
Gelatin type B powder (300 g Bloom) from
porcine skin was prepared from Sigma (USA).
PCL (Mn 80,000) and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDAC) were obtained
from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco
Modified Eagle’s Medium (DMEM), fetal
bovine serum (FBS), phosphate buffered saline
(PBS) and trypsin/EDTA were products of Gibco
(Invitrogen, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) and 4',6-diamidino-
2-phenylindole (DAPI) were purchased
from Promega (USA). All other chemicals and solvents
were obtained from Merck unless otherwise
stated and used without further modifications.

Mota A., Sahebghadam Lotfi A., Barzin J., Hatam M., Adibi B., Khalaj Z, & Massumi M. (2014). Human Bone Marrow Mesenchymal Stem Cell Behaviors on PCL/Gelatin Nanofibrous Scaffolds Modified with A Collagen IV-Derived RGD-Containing Peptide. Cell Journal (Yakhteh), 16(1), 1-10.

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Evaluation of Shikonin's Effect on Human Cervical Cancer Cells

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Hela and C33a, both human cervical cancer cell lines, were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, Grand Island, NY, USA) at 37°C in humidification environment encompassing 5% carbon dioxide (CO2). Shikonin was purchased from Meilun Biotechnology Co. (Dalian, China) and dissolved with dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was obtained from Promega (Madison, WI, USA). Cell Cycle staining kit was obtained from MultiSciences (Lianke) Biotechnology Co. (Hangzhou, China). Lipofectamine 3000 reagent was ordered from Life Technologies (AB &invitrogen, Carlsbad, CA, USA). Geneticin (G-418 Sulfate) was obtained from Life Technologies (Carlsbad, CA, USA). The D-luciferin was purchased from PerkinElmer (Waltham, MA, USA). Antibodies against Vimentin, E-cadherin, Snail and β-actin, and the secondary horseradish peroxidase (HRP)-labeled antibody were purchased from Cell Signaling Technology (CST; Beverly, MA, USA).

Tang Q., Liu L., Zhang H., Xiao J, & Hann S.S. (2020). Regulations of miR-183-5p and Snail-Mediated Shikonin-Reduced Epithelial-Mesenchymal Transition in Cervical Cancer Cells. Drug Design, Development and Therapy, 14, 577-589.

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Cytotoxicity Evaluation of Ouabain

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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Ouabain was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from (Promega Corporation, Madison, WI, USA).

Yang X.S., Xu Z.W., Yi T.L., Xu R.C., Li J., Zhang W.B., Zhang S., Sun H.T., Yu Z.Q., Xu H.X., Tu Y, & Cheng S.X. (2018). Ouabain suppresses the growth and migration abilities of glioma U-87MG cells through inhibiting the Akt/mTOR signaling pathway and downregulating the expression of HIF-1α. Molecular Medicine Reports, 17(4), 5595-5600.

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Osteosarcoma Cell Line Mitochondrial Study

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The human parental osteosarcoma cell line 143B and Rho°206 cells (mtDNA-depleted) were a gift from Professor Minxin Guan (Zhejiang University, Zhejiang, China). The cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% newborn calf serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin, 100 mg/ml streptomycin, 100 µg/ml bromodeoxy uridine and 50 µg/ml uridine (Sigma-Aldrich, St. Louis, MO, USA). Rhodamine 123 (Rh123) and dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) was purchased from Promega (Madison, WI, USA). We purchased cytochrome c (Cyt c) antibodies (12963S) from Cell Signaling Technology (CST; Beverly, MA, USA). Antibodies against β-actin (AA128) and the secondary horseradish peroxidase (HRP)-labeled antibodies (A0216), were purchased from Beyotime Biotechnology (Jiangsu, China).

Chen H., Wang J., Liu Z., Yang H., Zhu Y., Zhao M., Liu Y, & Yan M. (2016). Mitochondrial DNA depletion causes decreased ROS production and resistance to apoptosis. International Journal of Molecular Medicine, 38(4), 1039-1046.

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Rat Adrenal Pheochromocytoma Cell Study

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Rat adrenal pheochromocytoma (PC12) cells were used for this study. The cell line was obtained from ATCC. Bh and AA were purchased from Sigma (cat.# 122,228,198 and SH300341 respectively). RPMI, FBS and L-glutamic acid monosodium (lMSG) were purchased from Sigma-Aldrich Co. Lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagents were provided by Promega. Trypan Blue solution, L-glutamin, penicillin and streptomycin were obtained from HyClone U.S. MK801 blocker was purchased from Sigma-Aldrich, Germany. RNA Miniprep Kit was purchased from Promega, USA, and RT-PCR from Norgen Biotek, Canada. Acetylthiocholine iodide (ATCI), butyrylthiocholineiodide (BTCI) and all other reagents used for the current study were purchased from Sigma–Aldrich (USA).

Al Mughairbi F., Nawaz R., Khan F., Hassan A., Mahmood N., Ahmed H.T., Alshamali A., Ahmed S, & Bashir A. (2021). Neuroprotective effects of Bhilawanol and Anacardic acid during glutamate-induced neurotoxicity. Saudi Pharmaceutical Journal : SPJ, 29(9), 1043-1049.

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Evaluation of NSCLC Cell Viability

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A549 human NSCLC cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM-S to 80% confluence and then in serum-free DMEM for 24 h at 37°C in a 5% CO 2 atmosphere. Next, the tumor cells were cultured in 96-well plates (5,000 cells per well) in the presence of CM from CAFs or NFs for 72 h at 37°C in a 5% CO 2 atmosphere. Then, 15 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Promega Corporation, Madison, WI, USA) was added to each well, and the plate was incubated for 4 h at 37°C. Finally, 100 μL of solubilization/ stop solution (Promega) was added, and the absorbance was measured at 570 nm. Tumor cells were also cultured in serum-free DMEM as controls.

Negrete-Garcia M.C., Ramírez-Rodriguez S.L., Rangel-Escareño C., Muñoz-Montero S., Kelly-García J., Vázquez-Manríquez M.E., Santillán P., Ramírez M.M., Ramírez-Martínez G., Ramírez-Venegas A, & Ortiz-Quintero B. (2018). Deregulated MicroRNAs in Cancer-Associated Fibroblasts from Front Tumor Tissues of Lung Adenocarcinoma as Potential Predictors of Tumor Promotion. The Tohoku journal of experimental medicine, 246(2).

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